Short hairpin rnas for inhibition of gene expression

ABSTRACT

Methods, compositions, and kits that include small hairpin RNA (shRNA) useful for inhibition of gene expression, such as viral-mediated gene expression, are described.

This application claims the benefit of U.S. provisional application Ser.No. 61/105,606, filed Oct. 15, 2008, the entirety of which isincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OF DEVELOPMENT

This invention was made in part during work supported by NIH grantR44AI056611 (BHJ) from the National Institutes of Health. The governmentmay have certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to inhibition of viral gene expression, forexample, hepatitis C IRES-mediated gene expression, with short hairpinRNA (shRNA).

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is a cellular process that uses double strandedRNA (dsRNA) to target messenger RNA (mRNAs) for degradation andtranslation attenuation. The process is gene specific, refractory tosmall changes in target sequence, and amenable to multigene targeting.This phenomenon was first reported in plants in 1990 (Napoli, C., etal., Introduction of a chimeric chalcone synthase gene into petuniaresults in reversible co-suppression of homologous genes in trans. PlantCell, 1990, 2(4):279-289). It was later observed in other organismsincluding fungi and worms (Romano, N., et al., Quelling: transientinactivation of gene expression in Neurospora crassa by transformationwith homologous sequences. Mol. Microbiol., 1992, 6(22):3343-53).Mechanistically, long dsRNA can be cleaved into short interfering RNA(siRNA) duplexes by Dicer, a Type III RNase. Subsequently, these smallduplexes interact with the RNA Induced Silencing Complex (RISC), amultisubunit complex that contains both helicases and endonucleaseactivities that mediate degradation of homologous transcripts. Thediscovery that synthetic siRNAs of ˜19-29 by can effectively inhibitgene expression in mammalian cells and animals has led to a flurry ofactivity to develop these inhibitors as therapeutics (Elbashir, S. M.,et al., Duplexes of 21-nucleotide RNAs mediate RNA interference incultured mammalian cells. Nature, 2001, 411(6836):494-8). Recentdevelopment of advanced siRNA selection methods, includingalgorithm-based rational design selection, allows the researchers toselect potent siRNA duplexes by key sequence and thermodynamicparameters that are target sequence independent (Khvorova, A., et al.,Functional siRNAs and to miRNAs exhibit strand bias. Cell, 2003,115(1):209-216).

Small hairpin RNAs (shRNA) of 19-29 by that are chemically synthesizedor expressed from bacteriophage (e.g., T7, T3 or SP6) or mammalian (polIH such as U6 or H1 or pol II) promoters have also shown robustinhibition of target genes in mammalian cells. Furthermore, syntheticshRNA with its unimolecular structure has advantages in potency andsimplicity over two-strand-comprising siRNA, the latter requiring thecareful annealing of exact stoichiometric amounts of two separate strandwhich may have different purity profiles and off-target effects (Siolas,D., et al., Synthetic shRNAs as potent RNAi triggers. NatureBiotechnology, 2005, 23(2):227-231; Vlassov, A. V., et al., shRNAstargeting hepatitis C: effects of sequence and structural features, andcomparison with siRNA. Oligonucleotides, 2007, 17:223-236).

However, a number of challenges have arisen over the course of shRNAdesign and development. First, researchers have observed wide-rangingvariability in the level of silencing induced by different shRNA.Second, shRNA that varies in stem length, loop length and loop positionhas different knockdown capability (McManus, M. T., et al., Genesilencing using micro-RNA designed hairpins. RNA, 2002, 8:842-850;Harborth, J., et al., Sequence, chemical, and structural variatioin ofsmall interfering RNAs and short hairpin RNAs and the effect onmammalian gene silencing. Antisense And Nucleic Acid Drug Development.2003, 13:83-105; Li, L., et al., Defining the optimal parameters forhairpin-based knockdown constructs. RNA, 2007, 13:1765-1774; Vlassov, A.V., et al., shRNAs targeting hepatitis C: effects of sequence andstructural features, and comparison with siRNA. Oligonucleotides, 2007,17:223-236). Some shRNAs having a 19-nucleotide guide strand at the 5′end of the hairpin (left-hand loop) have been found to be moreefficacious than those having their guide strand at the 3′ end of thehairpin (right-hand loop) (Scaringe, S. US 2004/0058886 A1), while othershRNAs were reported to be more efficacious with the right-hand loopstructure (Vermeulen, et al. US 2006/0223777 A1). shRNAs with a 19-merguide strand at the 3′ end of the hairpin (right-hand loop) were foundto be more potent than 25- or 29-mer shRNAs (with right-hand loop).Previously available limited data suggested that the optimal loop sizefor a shRNA with a 19-bp stem with right-hand loop is larger than 4nucleotides and preferably 9 or 10 nucleotides. A third challenge isthat the mechanism of processing in the cytoplasm of synthetic shRNAs,especially 19-mer hairpins, is unknown. Unlike long dsRNAs and 29-mershRNAs, 19-mer shRNAs are not cleaved by Dicer (Siolas, D., et al.,Synthetic shRNAs as potent RNAi triggers. Nature Biotechnology, 2005,23(2):227-231). Therefore, it would be advantageous to have a reliabledesign for superior shRNAs and a better understanding of thestructure-activity relationship and processing of shRNAs to providehighly functional inhibitors.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions pertainingto shRNA, fractured shRNA, and T-shaped RNA for use in gene silencing.Accordingly, the present invention provides compositions, methods, andkits for increasing the efficiency of RNA interference.

In one aspect, the invention features a polynucleotide, e.g., a DNA oran RNA molecule, comprising a first sequence (e.g., a first RNAsequence), e.g., a guide strand, consisting of 15 nucleotides to 30nucleotides, wherein the first sequence is at least partiallycomplementary to a target nucleotide sequence; a second sequence (e.g.,a second RNA sequence), e.g., a passenger strand, comprising a sequencethat is at least partially complementary to at least a portion of thefirst sequence, the second sequence having a length of between 10nucleotides and 1 fewer nucleotide than the length of the firstsequence; optionally a loop sequence positioned between the firstsequence and the second sequence, the loop consisting of 1 to 100nucleotides; and optionally a nucleotide overhang consisting of 1 to 2nucleotides.

In another aspect, the invention features a polynucleotide, e.g., a DNAor an RNA molecule, comprising a first sequence (e.g., a first RNAsequence), e.g., a guide strand, consisting of 16 nucleotides to 30nucleotides, wherein the first sequence is at least partiallycomplementary to a target nucleotide sequence; a second sequence (e.g.,a second RNA sequence), e.g., a passenger strand, comprising a sequencethat is at least partially complementary to at least a portion of thefirst sequence, the second sequence having at least one more nucleotidethan the first sequence, and the second sequence having no more than 100nucleotides; optionally a loop sequence positioned between the firstsequence and the second sequence, the loop consisting of 1 to 100nucleotides; and optionally a nucleotide overhang consisting of 1 to 2nucleotides.

In another apect, the invention features a polynucleotide, e.g., a DNAor an RNA molecule, comprising a first sequence (e.g., a first RNAsequence), e.g., a guide strand, consisting of 16 nucleotides to 18nucleotides, wherein the first sequence is at least partiallycomplementary to a target nucleotide sequence; a second sequence (e.g.,a second RNA sequence), e.g., a passenger strand, comprising a sequencethat is at least partially complementary to at least a portion of thefirst sequence, the second sequence having the same number ofnucleotides as the first sequence; optionally a loop sequence positionedbetween the first sequence and the second sequence, the loop consistingof 1 to 2 nucleotides; and optionally a nucleotide overhang consistingof 1 to 2 nucleotides.

In another aspect, the invention features a polynucleotide, e.g., a DNAor an RNA molecule, comprising a first sequence (e.g., a first RNAsequence), e.g., a guide strand, consisting of 19 nucleotides, whereinthe first sequence is at least partially complementary to a targetnucleotide sequence; a second sequence (e.g., a second RNA sequence),e.g., a passenger strand, comprising a sequence that is at leastpartially complementary to at least a portion of the first sequence, thesecond sequence consisting of 17 nucleotides or 18 nucleotides;optionally a loop sequence positioned between the first sequence and thesecond sequence, the loop consisting of 1 to 100 nucleotides; andoptionally a nucleotide overhang consisting of 1 to 2 nucleotides.

In another aspect, the invention features a polynucleotide, e.g., a DNAor an RNA molecule, comprising a first sequence (e.g., a first RNAsequence), e.g., a guide strand, consisting of 15 nucleotides to 30nucleotides, wherein the first sequence is at least partiallycomplementary to a target nucleotide sequence; a second sequence (e.g.,a second RNA sequence), e.g., a passenger strand, comprising a sequencethat is at least partially complementary to at least a portion of thefirst sequence, the second sequence having the same or fewer number ofnucleotides as the first sequence; optionally a loop sequence positionedbetween the first sequence and the second sequence, the loop consistingof 1 nucleotide; and optionally a nucleotide overhang consisting of 1 to2 nucleotides.

In another aspect, the invention features an RNA molecule comprising afirst RNA sequence, e.g., a guide strand, consisting of about 5nucleotides to about 15 nucleotides, wherein the first sequence is atleast partially complementary to a target nucleotide sequence; a secondRNA sequence, e.g., a passenger strand, capable of forming a hairpinstructure with the first sequence, the hairpin structure comprising fromabout 23 nucleotides to about 100 nucleotides, and the second sequencecomprising: a first region having at least 80% complementarity with thefirst sequence and being capable of forming a first duplex region offewer than 19 base pairs with the first sequence; a second regioncoupled to the first region; a third region coupled to the secondregion; and a fourth region coupled to the third region and having atleast 80% complementarity with the second region, the fourth regionbeing capable of forming a second duplex region with the second regionsuch that the third region forms a loop adjacent to the second duplexregion and the sum of the lengths of the first duplex region and thesecond duplex region is less than 23 base pairs; and optionally, anoverhang on the 5′ or 3′ end of the RNA molecule of fewer than about 6nucleotides.

In another aspect, the invention features an RNA molecule comprising anRNA sequence of any one of SEQ ID NOs: 1-39.

In some embodiments, the third region forms a loop that includesnucleotides having the sequence of 5′-UU-3′. Further, the second strandcan include a part of passenger strand with a 5′ phosphate group. In oneembodiment, the second strand with the 5′ phosphate group is adjacent tothe fourth region of the second strand. In another embodiment, thesecond strand with the 5′ phosphate group is not adjacent to the fourthregion of the second strand.

In another aspect, the invention features a T-shaped RNA molecule,comprising a first RNA sequence, e.g., a guide strand, that is at leastpartially complementary to a target nucleotide sequence, a second RNAsequence, e.g., a passenger strand, and a third RNA sequence, whereinthe first sequence comprises a first region having from about 18nucleotides to about 29 nucleotides at least 80% complementary to afirst region of the second sequence; the first sequence furthercomprises a second region having about 4 nucleotides to about 10nucleotides at least about 90% complementary to a first region of thethird sequence; the second sequence comprises a first region having fromabout 18 nucleotides to about 29 nucleotides at least about 80%complementary to the first region of the first sequence; the secondsequence further comprises a second region having about 4 nucleotides toabout 10 nucleotides at least about 90% complementary to a second regionof the third sequence, wherein the second region of the third sequenceis 3′ adjacent to the first region of the third sequence; andoptionally, an overhang on the 5′ or 3′ end of the RNA molecule of fewerthan about 6 nucleotides.

In some embodiments of any of the aspects described herein, apolynucleotide, e.g., a DNA or RNA molecule described herein, caninclude from about 30 nucleotides to about 84 nucleotides.

In some embodiments of any of the aspects described herein, apolynucleotide, e.g., a DNA or RNA molecule described herein, includes asecond sequence that comprises a sequence that is at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, orabout 100% complementary to at least a portion of the first sequence. Inparticular embodiments, the second sequence comprises a sequence, e.g.,at the 3′ end of the second sequence, that is at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, orabout 100% complementary to a portion at the 5′ end of the firstsequence.

In some embodiments of any of the aspects described herein, apolynucleotide, e.g., a DNA or RNA molecule described herein, includesat least one modification, e.g., chemical modification described herein.In some embodiments, about 20% to about 100%, e.g., about 40% to about90%, of the nucleotides in an RNA molecule described herein arechemically modified. In one embodiment, about 20% to about 100% of theUs and Cs in an RNA molecule described herein are 2′ O-methyl modifiedor 2′ F.

In some embodiments of any of the aspects described herein, apolynucleotide, e.g., a DNA or RNA molecule described herein, includes aloop that includes at least one non-nucleotide molecule.

In some embodiments of any of the aspects described herein, apolynucleotide, e.g., a DNA or RNA molecule described herein, includesan overhang at the 3′ end or the 5′ end of the molecule. For example,the overhang is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

In some embodiments of any of the aspects described herein, apolynucleotide, e.g., a DNA or RNA molecule described herein, includes 1or 2 mismatches between the first sequence and the second sequence. Inone embodiment, a mismatch exists at position 11, 12, 13, 14, 15, 16, or17 on the guide strand counting from the 5′ end and the oppositenucleotide in the passenger strand, preferably at position 14 on theguide strand counting from the 5′ end and the opposite nucleotide in thepassenger strand.

In some embodiments of any of the aspects described herein, the firstsequence is 3′ to the second sequence. In other embodiments, the firstsequence is 5′ to the second sequence.

In some embodiments of any of the aspects described herein, an RNAmolecule described herein further includes at least one conjugate moietyattached to the loop region or 3′ terminus of the RNA molecule via alinker. The conjugate moiety can be a steroid molecule, a vitamin, apeptide, galactose and derivatives thereof, or a protein. In someembodiments, the conjugate moiety is a steroid molecule selected fromthe group consisting of cholesterol, cholestanol, stigmasterol, cholanicacid, and ergosterol. In other embodiments, the conjugate moiety ischolesterol, and the linker is a C5 linker molecule. In yet otherembodiments, the conjugate moiety is vitamin E.

In other embodiments, an RNA molecule described herein further includesat least one detectable label attached to the loop region or 3′ terminusof the RNA molecule. In some embodiments, the detectable label is a dyemolecule. In some embodiments, the RNA molecule can include both aconjugate moiety and a detectable label.

In some embodiments, an RNA molecule described herein is capable ofinhibiting expression of the target nucleotide sequence, e.g., a viralsequence. In some embodiments, the viral sequence is a hepatitis C viralsequence. In certain embodiments, the target nucleotide sequence is asequence within the internal ribosome entry site (IRES) sequence ofhepatitis C virus.

In another aspect, the invention features a DNA sequence comprising asequence encoding an RNA molecule described herein, or an expressionvector comprising a DNA sequence comprising a sequence encoding an RNAmolecule described herein. In some embodiments, the vector is aretroviral vector.

In another aspect, the invention features a composition comprising anRNA molecule described herein, and a pharmaceutically acceptableexcipient. In another aspect, the invention features a compositioncomprising a vector comprising a sequence encoding an RNA moleculedescribed herein.

In another aspect, the invention features a method of inhibiting theexpression or activity of a gene, the method comprising contacting acell that expresses the gene with an RNA molecule described herein,wherein the first RNA sequence is at least partially complementary to atarget nucleotide sequence encoded by at least a portion of the gene.

In another aspect, the invention features a method of inhibitingexpression or activity of a hepatitis C virus, the method comprisingcontacting a cell that expresses a hepatitis C virus with an RNAmolecule described herein, wherein the first RNA sequence is at leastpartially complementary to a hepatitis C viral sequence.

In any of these aspects, the cell can be in a mammal, e.g., a human or anon-human primate.

In certain embodiments, an RNA molecule described herein does not inducean IFN response.

In another aspect, the invention features a kit comprising a container,the container comprising an RNA molecule described herein; andoptionally a reduced serum tissue culture medium.

The following figures are presented for the purpose of illustrationonly, and are not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the structure of embodiments of hairpin containingvarious configurations.

FIG. 2 depicts the structure of T-shaped RNA molecule and multiplicationof T-shaped RNA molecule by having a long third strand with repeatedsequence complementary to the second region of first and second strands.

FIGS. 3A-3C compare the dose response of shRNAs having 5′-passengerstrand and that having 5′-guide strand. The shRNAs were synthesized byIDT and reconstituted with siRNA buffer (Dharmacon, containing 20 mMKCl, 6 mM HEPES-KOH pH 7.5, 0.2 mM MgCl₂). 293FT cells (23,000 cells perwell in 96-well plate) were cotransfected with shRNAs and target DNAplasmid in which firefly luciferase expression is dependent on the HCVIRES (IRES-fLuc). Lipofectamine™ 2000 (Invitrogen) was used astransfection reagent. Forty-eight hours later, the cells were lysed andfirefly luciferase activity was measured by a luminometer. All data arethe results of individual, independent experiments performed intriplicate. FIGS. 3D-3F compare the same set of shRNAs for theirinternal stabilities calculated by Oligo 4.0. Results demonstrate thatshRNAs with 5′-guide strand has enhanced functionality compared to thosewith 5′-passenger strand. The extent of enhancement issequence-dependent and the differences in the internal stability of the5′ termini make an important contribution.

FIG. 4 depicts the dose responses of shRNAs with various loop length,sequence and closing base pairs. The experiments were done as describedin FIG. 3. FIG. 4A compares the potency of shRNA with 10-, 5-, 2-, and1-nucleotide (nt) loop. The sequences of the duplex regions are the sameamong these shRNAs and siRNA 19-3. The results indicate that shRNAs with5′-guide strands have higher potencies when the loop size is very small(1- or 2-nt) than when it is larger (5- or 10-nt). The loop sequencelikely doesn't affect the shRNA activity since three different loopsequences were used. FIG. 4B compares the activity of shRNAs with U:Aand C:G base pairs immediately before the loop. The sequences of theloop (in underline) and the adjacent base pair are shown in parentheses.The sequence of the duplex region is the same among these shRNAs. Thecomparison indicates that when the loop is 2-nt in length, the shRNAhaving a C:G base pair adjacent to the loop gives better potency thanthat with an adjacent U:A pair. FIG. 4C shows that the UU-ribonucleotidesequence in the loop of shRNA can be replaced with aTT-deoxyribonucleotide sequence without affecting the efficacy of theshRNA. The sequence of the duplex is the same for both of these shRNAsand siRNA 19-3.

FIGS. 5 depicts the dose responses of shRNAs with various duplex lengthsand 3′ overhangs. The experiments were done as described in FIG. 3. FIG.5A compares the activity of shRNAs with and without 3′ overhang. Theresult indicates that the shRNAs with blunt end or with 3′-TT overhanghave very similar potency as the corresponding shRNA with 3′-UUoverhang. FIG. 5B compares the potency of shRNA with various lengths ofduplex or passenger strand. g/p values represent the lengths of guidestrand/passenger strands. The comparison suggests that shorteningpassenger strand to 17- or 16-nt while maintaining the length of guidestrand at 19-nt significantly reduces gene silencing activity (SG115 andSG116). However, shortening both passenger and guide strands to 18-nt inlength did not have a significant impact on potency (SG117). FIG. 5Cdepicts the activity of shRNAs with even shorter duplex lengths. TheshRNAs with 17 or 16 base pairs in the duplex (SG117 and SG119) havevery similar efficacy. Although the shRNA with 11 base pairs in theduplex (SG120) shows a significant loss of silencing activity, the IC₅₀of this molecule is still in the subnanomolar range.

FIGS. 6 depicts the dose responses of shRNAs with various short stemlengths. The experiments were done as described in FIG. 3. FIG. 6Acompares the activity of shRNAs varying lengths of both guide andpassenger strands. The result shows that the potency of shRNAs decreasessignificantly when shRNAs with duplex lengths of 14-base pairs or lesswere used (SG132 and SG120). FIG. 6B depicts the activity of shRNAs with10- to 13-nt passenger strands while maintaining the length of guidestrand at 19-nt. The result indicates that the activity of shRNArequires the hairpin containing a minimum length of 11-nt for thepassenger strand.

FIGS. 7 depicts the potencies of various structured shRNAs againstdifferent targets. The experiments were done as described in FIG. 3.FIGS. 7A and 7B show the activity of shRNAs against the same target assi72 and si74, respectively, but differ in structures. The resultsindicate that 3′-UU overhang is not essential for the activity of shRNA.Shortening the duplex region could partially reduce the target geneknockdown activity.

FIG. 8 depict the dose responses of shRNAs with single mismatch atdifferent position of the duplex region. FIG. 8A shows a similaractivity between shRNA with (SG110) and without (SG142) a singlemismatch at position 6 from 5′ end of the passenger strand (oppositeposition 14 from 5′ end of the guide strand). FIG. 8B shows a similaractivity among shRNAs without (SG142) and with a single mismatch atposition 6 (SG126), 7 (SG127), 5 (SG128), and 4 (SG129) from 5′ end ofthe passenger strand respectively (opposite position 14, 13, 15 and 16from 5′ end of the guide strand). This lack of activity loss with singlemismatch appears to be unrelated to the type of mismatch (e.g., U-U(SG110)=U-C (SG126)) suggesting that this trait is sequence independent.All the shRNAs have 5′-UU-3′ loop and 3′-UU overhang.

FIG. 9 depicts that the target inhibition of shRNAs are not due to dimeror oligomer formation. FIG. 9A compares the target inhibition of shRNAwith and without heating and snap-cooling. Although slightly lower thanthe shRNAs with multiple species, the single species of shRNA monomersobtained by heating and snap-cooling show high activity in target genesilencing irrespective of sequence or length tested. FIG. 913 shows themonomer, dimer and oligomer of shRNA in 10% naïve polyacrylamide gelwith and without heating (95° C. for 4 minutes) and snap-cooling ofshRNA in ice-water bath. The gel was stained with SYBR Gold(Invitrogen).

FIG. 10 depicts the different behaviors of shRNAs in monomer and mixedspecies. FIG. 10A shows that SG119 has higher silencing activity whenmajority is monomer whereas SG120 has higher silencing activity whenmajority is dimer. FIG. 10B shows the monomer, dimer and oligomer ofshRNA in 10% naïve polyacrylamide gel with and without heating (95° C.for 4 minutes) and snap-cooling of shRNA in ice-water bath.

FIG. 11 depicts the potency of an shRNA with and without a nick and of aT-shaped RNA. The experiments were done as described in FIG. 3. Theconcentrations of shRNA used in these experiments include 0.3 nM, 0.1nM. 0.03 nM. 0.01 nM and 0.003 nM. Only 2 concentrations (0.3 and 0.03nM) were tested with SG102. SG68L, SG146 and SG142 have the same duplexsequence with 3′-UU overhang. SG68L and SG146 have 5′-CAAUA-3′ loopwhereas SG142 has 5′-UU-3′ loop. SG146 is composed of two RNA moleculesthat were annealed together according to Dharmacon's siRNA annealinginstruction. The first molecule contains guide strand, loop and4-nucleotide passenger strand; the second molecule contains 3′ end of15-nucleotide passenger strand and 3′ overhang. SG102 is composed of 3RNA molecules (FIG. 2A) with guide and passenger strands in the firstregion of the first and second strands. The third strand iscomplementary to the second regions of the first and second strands.

FIG. 12 depicts the potency of shRNAs in the hepatocarcinoma cell lineHuh7. Consistent with the results in 293FT cells, SG68L is more potentthan SG68. The negative control, SG101, did not reduce the fireflyluciferase gene expression at any given concentrations.

FIG. 13 shows that the inhibitory activity of several highly potentshRNAs is not due to an IFN response or cytotoxicity. FIG. 13A depictsthe expression of the IFN-responsive gene OAS1 in human peripheral bloodmononuclear cells (PBMC) after shRNA transfection. FIG. 1313 depicts the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)incorporation in human hepatocarcinoma cell line (Huh7) after shRNAtransfection. Together, these panels of FIGS. 13A and 13B indicate thatno IFN response or cytotoxicity were induced by these shRNAs.

FIG. 14 shows that shRNAs with stem lengths of 19 or fewer base pairsare substrates for recombinant Dicer. The products of treatment by Dicerare analyzed by non-denaturing (FIG. 14A) and denaturing (FIG. 14B)PAGE.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein, including GenBankdatabase sequences, are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Embodiments of the present disclosure are directed to compositions andmethods for performing shRNA-induced gene silencing. Through the use ofshRNA, modified shRNA, fractured shRNA, modified fractured shRNA,T-shaped RNA and derivatives thereof, the efficiency of RNA interferencemay be improved. Accordingly, the present disclosure providescompositions, methods, and kits for increasing the functionality ofshRNA. Preferably, the disclosure provides compositions and methods forimproving the functionality of shRNA for inhibiting viral geneexpression and/or treating a viral infection in a mammal, such as ahuman. In some embodiments, the shRNA constructs described hereininhibit gene expression of a virus by inducing cleavage of viralpolynucleotide sequences within or near the target sequence that isrecognized by the guide strand sequence of the shRNA.

The phrase “short hairpin RNA” and the term “shRNA”, as used herein,refer to a unimolecular RNA that is capable of performing RNAi and thathas a passenger strand, a loop, and a guide strand. Preferably, thepassenger and guide strands are substantially complementary to eachother. The guide strand can be about 16 to about 29 nucleotides inlength, and more preferably 18 to 19 nucleotides in length. Thepassenger strand can be about 11 to about 29 nucleotides in length, andmore preferably 17 to 19 nucleotides in length. The guide strand cancontain at least 17 bases that are complementary to a target mRNA. Insome embodiments, the guide strand that is complementary to the targetcan contain mismatches. The sequence can be varied to target one or moregenetic variants or phenotypes of a target, e.g., a viral target, byaltering the targeting sequence to be complementary to the sequence ofthe genetic variant or phenotype. In some embodiments, a sequence cantarget multiple viral strains, e.g., of HCV, although the sequence candiffer from the target of a strain by at least one nucleotide (e.g.,one, two, or three nucleotides) of a targeting sequence. An shRNA mayhave a loop as long as, for example, 0 to about 24 nucleotides inlength, preferably 0 to about 10 nucleotides in length, and morepreferably 2 nucleotides in length. The sequence of the loop can includenucleotide residues unrelated to the target. In one particularlypreferred embodiment, the loop is 5′-UU-3′. In some embodiments it mayinclude non-nucleotides moieties. Yet in other embodiments, the loopdoes not include any non-nucleotides moieties. Optionally, the shRNA canhave an overhang region of 2 bases on 3′ end of the molecule. The shRNAcan also comprise RNAs with stem-loop structures that contain mismatchesand/or bulges. The passenger strand that is homologous to the target candiffer at about 0 to about 5 sites by having mismatches, insertions, ordeletions of from about 1 to about 5 nucleotides, as is the case, forexample, with naturally occurring microRNAs. RNAs that comprise any ofthe above structures can include structures where the loops comprisenucleotides, non-nucleotides, or combinations of nucleotides andnon-nucleotides. Within any shRNA, preferably a plurality and morepreferably all nucleotides are ribonucleotides.

In some embodiments, an shRNA described herein optionally includes atleast one conjugate moiety.

The phrase “fractured shRNA”, as used herein, refers to a short hairpinRNA that comprises two or more distinct strands. Such molecules can beorganized in a variety of fashions (e.g.,5′-passenger-fracture-passenger-loop-guide,5′-guide-loop-passenger-fracture-passenger) and the fracture in themolecule can comprise a nick, a nick bordered by one or more unpairednucleotides, or a gap.

The phrase “T-shaped RNA” as used herein, refers to a T-shaped RNA thatcomprises at least three separate strands: a first strand, a secondstrand and a third strand. The first and second strands both comprisetwo regions (1 and II) as follows: the first region of the first strandis capable annealing or hybridizing with the first region of the secondstrand; the first region of one of the strand can contain at least 17bases that are complementary to a target mRNA; the first region of bothstrands can be about 17 to 29 nucleotides in length and preferably 22nucleotides in length; the second region of the first strand can annealor hybridize with part of the third strand; the second region of thesecond strand can anneal or hybridize with part of the third strand thatis adjacent and 3′ to the part that hybridizes with the second region ofthe first strand; the second regions of the first and second strands arenot complementary to each other and can be about 4 to about 10nucleotides in length, preferably 8 nucleotides in length; the thirdstrand can be at least about 8 nucleotides in length; the third strandmay have multiple sequences that are complementary to the second regionsof her pairs of first and second strands so that multiple duplexescomprised of first and second strands can be annealed to the thirdstrand; optionally, there is an overhang region of 2 bases on 3′ end ofthe second strand.

The shRNAs, fractured shRNAs, and T-shaped RNAs described herein can beuseful in implementing gene silencing. Also, they may be preferred overduplexes having lengths that are similar or equivalent to the length ofthe stem of the hairpin in some instances, due to the fact that theshRNAs described herein can be more efficient in RNA interference andless likely to induce cellular stress and/or toxicity.

Additionally, the phrase “short hairpin RNA” and the term “shRNA”include nucleic acids that also contain moieties other thanribonucleotide moieties, including, but not limited to, modifiednucleotides, modified internucleotide linkages, non-nucleotides,deoxynucleotides and analogs of the nucleotides mentioned thereof.

The term “siRNA”, as used herein, refers to an RNA molecule comprising adouble stranded region and a 3′ overhang of nonhomologous residues ateach end. The double stranded region is typically about 18 to about 30nucleotides in length, and the overhang may be of any length ofnonhomologous residues, but a 2 nucleotide overhang is preferred.

The phrase “guide strand”, as used herein, refers to a polynucleotide orregion of a polynucleotide that is substantially complementary (e.g.,80% or more) or 100% complementary to a target nucleic acid of interest.The guide strand of a shRNA is also at least substantially complementaryto its passenger strand. A guide strand can be composed of apolynucleotide region that is RNA, DNA or chimeric RNA/DNA. Anynucleotide within a guide strand can be modified by includingsubstituents coupled thereto, such as in a 2′ modification. The guidestrand can also be modified with a diverse group of small moleculesand/or conjugates. For example, a guide strand may be complementary, inwhole or in part, to a molecule of messenger RNA, an RNA sequence thatis not mRNA (e.g., tRNA, rRNA, hnRNA, negative and positive strandedviral RNA and its complementary RNA) or a sequence of DNA that is eithercoding or non-coding.

The guide strand may be part of a larger strand that comprisesnucleotides other than guide strand nucleotides. For example, in thecase of a T-shaped RNA structure the first or second strand can containa guide strand and additional nucleotides that are complementary to thethird strand, but not complementary to the target. In the case of afractured hairpin, the guide strand can be part of a strand that alsocomprises loop region and a third region that is complementary to partof guide strand.

The phrase “passenger strand”, as used herein, refers to apolynucleotide or region that has the same nucleotide sequence, in wholeor in part, as a target nucleic acid such as a messenger RNA or asequence of DNA. When a sequence is provided, by convention, unlessotherwise indicated, it is the passenger strand (or region), and thepresence of the complementary guide strand (or region) is implicit.

The term “complementary”, as used herein, refers to the ability ofpolynucleotides to form base pairs with one another. Base pairs aretypically formed by hydrogen bonds between nucleotide units inantiparallel polynucleotide, strands or regions. Complementarypolynucleotide strands or regions can base pair in the Watson-Crickmanner (e.g., A to T, A to U, C to G), or in any other manner thatallows for the formation of stable duplexes.

“Perfect complementarity” or “100% complementarity”, as used herein,refers to the situation in which each nuclotide unit of onepolynucleotide strand or regioin can hydrogen bond with each nucleotideunit of a second polynucleotide strand or region. Less than perfectcomplementarity refers to the situation in which some, but not all,nucleotide units of two strands or two regions can hydrogen bond witheach other. For example, for two 19-mers, if 17 base pairs on eachstrand or each region can hydrogen bond with each other, thepolynucleotide strands exhibit 89.5% complementarity. Substantialcomplementarity refers to polynucleotide strands or regions exhibiting80% or greater complementarity.

The term “deoxynucleotide”, as used helrein, refers to a nucleotide orpolynucleotide lacking an OH group at the 2′ or 3′ position of a sugarmoiety with appropriate bonding and/or 2′,3′ terminal dideoxy, insteadhaving a hydrogen bonded to the 2′ and/or 3′ carbon.

The terms “deoxyribonucleotide” and “DNA”, as used herein, refer to anucleotide or polynucleotide comprising at least one ribosyl moiety thathas an H at its 2′ position of a ribosyl moiety instead of an OH.

The term “mismatch”, as used herein, includes situations in whichWatson-Crick base pairing does not take place between a nucleotide of aguide strand and a nucleotide of a passenger strand, where thenucleotides are flanked by a duplex comprising base pairs in the 5′direction of the mismatch beginning directly after (in the 5′ direction)the mismatched position and in the 3′ direction of the mismatchbeginning directly after (in the 3′ direction) the mismatched position.Examples of mismatches include, without limitation, an A across from aG, a C across from an A, a U across from a C, an A across from an A, a Gacross from a G, a C across from a C, and so on. Mismatches also includean abasic residue across from a nucleotide or modified nucleotide, anacyclic residue across from a nucleotide or modified nucleotide, a gap,or an unpaired loop. In its broadest sense, a mismatch includes anyalteration at a given position that decreases the thermodynamicstability at or in the vicinity of the position where the alterationappears, such that the thermodynamic stability of the duplex at theparticular position is less than the thermodynamic stability of aWatson-Crick base pair at that position. Preferred mismatches include aG across from an A, and an A across from a C. A particularly preferredmismatch comprises an A across from an A, G across from a G, C acrossfrom a C, and U across from a U.

The phrase “RISC” and “RNA induced silencing complex” are usedinterchangeably herein, and represent a complex of proteins that mediateRNAi (see, e.g., Hutvagner, G. FEBS Letters, 2005 579(26):5850-7).

The phrase “RNA interference” and the term “RNAi” are usedinterchangeably herein, and refer to the process by which a single,double, or T-shaped molecule (e.g., an siRNA, an shRNA, an miRNA, apiRNA) exerts an effect on a biological process by interacting with oneor more components of the RNAi pathway including, but not limited to,Drosha, DISC, Dicer, etc. The process includes, but is not limited to,gene silencing by degrading mRNA; attenuating translation, interactionswith tRNA, rRNA, hnRNA, cDNA and genomic DNA; and inhibiting as well asmethylating DNA with ancillary proteins. In addition, molecules thatmodulate RNAi (e.g., siRNA, piRNA, or miRNA inhibitors) are included inthe list of molecules that enhance the RNAi pathway (see, e.g., Tomari,Y. et al. Genes Dev. 2005, 19(5):517-29).

The phrase “silencing”, as used herein, means an RNAi-mediated reductionin gene expression that can be measured by any number of methodsincluding reporter methods such as for example luciferase reporterassay, PCR-based methods, Northern blot analysis, Branched DNA, westernblot analysis, and other art recognized techniques.

The term “alkyl”, as used herein, refers to a hydrocarbyl moiety thatcan be saturated or unsaturated, and substituted or unsubstituted. Itmay comprise moieties that are linear, branched, cyclic and/orheterocyclic, and contain functional groups such as ethers, ketones,aldehydes, carboxylates, etc. Unless otherwise specified, alkyl groupsare not cyclic, heterocyclic, or comprise functional groups.

Exemplary alkyl groups include, but are not limited to, substituted andunsubstituted groups of methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicoyl andalkyl groups of higher number of carbons, as well as 2-methylpropyl,2-methyl-4-ethylbutyl, 2,4-diethylpropyl, 3-propylbutyl,2,8-dibutyldecyl, 6,6-dimethyloctyl, 6-propyl-6-butyloctyl,2-methylbutyl, 2-methylpentyl, 3-methylpentyl, nad 2-ethylhexyl. Theterm alkyl also encompasses alkenyl groups, such as vinyl, allyl,aralkyl and alkynyl groups. Unless otherwise specified, alkyl groups arenot substituted.

Substitutions within an alkyl group, when specified as present, caninclude any atom or group that can be tolerated in the alkyl moiety,including but not limited to halogens, sulfurs, thiols, thioethers,thioesters, amines (primary, secondary, or tertiary), amides, ethers,esters, alcohols and oxygen. The alkyl groups can by way of example alsocomprise modifications such as azo groups, keto groups, aldehyde groups,carboxyl groups, nitro, nitroso or nitrile groups, heterocycles such asimidazole, hydrazine or hydroxylamino groups, isocyanate or cyanategroups, and sulfur containing groups such as sulfoxide, sulfone,sulfide, and disulfide. Unless otherwise specified, alkyl groups do notcomprise halogens, sulfurs, thiols, thioethers, thioesters, amines,amides, ethers, esters, alcohols, oxygen, or the modifications listedabove.

Further, alkyl groups may also contain hetero substitutions, which aresubstitutions of carbon atoms, by for example, nitrogen, oxygen orsulfur. Heterocyclic substitutions refer to alkyl rings having one ormore heteroatoms. Examples of heterocyclic moieties include but are notlimited to morpholino, imidazole, and pyrrolidino. Unless otherwisespecified, alkyl groups do not contain hetero substitutions or alkylrings with one or more heteroatoms (i.e., heterocyclic substitutions).

The preferred alkyl group for a 2′ modification is a methyl group withan O-linkage to the 2′ carbon of a ribosyl moiety, i.e., a 2′ O-alkylthat comprises a 2′-O-methyl group.

The phrase “2′-O-alkyl modified nucleotide”, as used herein, refers to anuclotide unit having a sugar moiety, for example a deoxyribosyl moietythat is modified at the 2′ position such that an oxygen atom is attachedboth to the carbon atom located at the 2′ position of the sugar and toan alkyl group. In various embodiments, the alkyl moiety consistsessentially of carbons and hydrogens. A particularly preferredembodiment is one wherein the alkyl moiety is methyl.

As used herein, the term “2′ carbon modification” refers to a nucleotideunit having a sugar moiety, for example a moiety that is modified at the2′ position of the sugar subunit. A “2′-O-alkyl modified nucleotide” ismodified at this position such that an oxygen atom is attached both tothe carbon atom located at the 2′ position of the sugar and to an alkylgroup, e.g., 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O -isopropyl2′-O-butyl, 2′-O-isobutyl, 2′-O-ethyl-O-methyl(—OCH₂CH₂OCH₃), and2′-O-ethyl-OH (—OCH₂CH₂OH). A “2′ carbon passenger strand modification”,as used herein, refers to a modification at the 2′ carbon position of anucleotide on the passenger strand. A “2′ carbon guide strandmodification”, as used herein, refers to a modification at the 2′ carbonposition of a nucleotide on the guide strand.

The term “nucleotide”, as used herein, refers to a ribonucleotide or adeoxyribonucleotide or modified from thereof, as well as an analogthereof. Nucleotides include species that comprise purines, e.g.,adenine, hypoxanthine, guanine, and their derivatives and analogs, aswell as pyrimidines, e.g., cytosine, uracil, thymine, and theirderivatives and analogs. Preferably, a “nucleotide” comprises acytosine, uracil, thymine, adenine, or guanine moiety.

Nucleotide analogs include nucleotides having modifications in thechemical structure of the base, sugar and/or phosphate, including, butnot limited to, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at cytosine exocyclic amines, andsubstitution of 5-bromo-uracil; and 2′-position sugar modifications,including but not limited to, sugar-modified ribonucleotides in whichthe 2′-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH₂,NHR, NR₂, or CN, wherein R is an alkyl moiety as defined herein.Nucleotide analogs also include nucleotides with bases such as inosine,queuosine, xanthine, sugars such as 2′-methyl ribose, non-naturalphophodiester linkages such as methylphosphonates, phophorothioates andpeptides.

Modified bases refer to nucleotide bases such as, for example, adenine,guanine, cytosine, thymine, and uracil, xanthine, inosine, and queuosinethat have been modified by the replacement or addition of one or moreatoms or groups. Some examples of types of modifications includenucleotides that are modified with respect to the base moieties, includebut are not limited to, alkylated, halogenated, thiolated, aminated,amidated, or acetylated bases, in various combinations. More specificmodified bases include, for example, 5-propynyluridine,5-propynylcytidine, 6-methyladenine, 6-methylguanine,N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2′-aminoadenine,1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine andother nucleotides having a modification at the 5 position,5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosien,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any O- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides. Modified nucleotides also includethose nucleotides that are modified with respect to the sugar moiety, aswell as nucleotides having sugars or analogs thereof that are notribosyl. For example, the sugar moieties may be, or be based on,mannoses, arabinoses, glucopyranoses, galactopyranoses, 4-thioribose,and other sugars, heterocycles, or carbocycles. The term nucleotideanalog also includes what are known in the art as universal bases. Byway of example, universal bases include but are not limited to3-nitropyrrole, 5-nitroindole, or nebularine.

Further, the term nucleotide analog also includes those species thathave a detectable label, such as, for example, a radioactive orfluorescent moiety, or mass label attached to the nucleotide.

The term “overhang”, as used herein, refers to terminal non-base pairingnucleotide(s) resulting from one strand or region extending beyond theterminus of the complementary strand to which the first strand or regionforms a duplex. One or more polynucleotides that are capable of forminga duplex through hydrogen bonding can have overhangs. Thesingle-stranded region extending beyond the 3′ end of the duplex isreferred to as an overhang.

The term “ribonucleotide” and the phrase “ribonucleic acid” (RNA), asused herein, refer to a modified or unmodified nucleotide orpolynucleotide comprising at least one ribonucleotide unit. Aribonucleotide unit comprises an oxygen attached to the 2′ position of aribosyl moiety having a nitrogenous base attached in N-glycosidiclinkage at the 1′ position of a ribosyl moiety, and a moiety that eitherallows for linkage to another nucleotide or precludes linkage.

The phrase “pharmaceutically acceptable carrier”, as used herein, meansa pharmaceutically acceptable salt, solvent, suspending agent or vehiclefor delivering a composition of the present disclosure to the animal orhuman. The carrier may be liquid, semisolid or solid, and is oftensynonymously used with diluent, excipient or salt. The phrase“pharmaceutically acceptable” means that an ingredient, excipient,carrier, diluent or component disclosed is one that is suitable for usewith humans and/or animals without undue adverse side effects (such astoxicity, irritation, and allergic response) commensurate with areasonable benefit/risk ratio. See Remington's Pharmaceutical Sciences16^(th) edition, Osol, A. Ed (1980).

The term “about” is used herein to mean a value ±20% of a givennumerical value. Thus, “about 60%” means a value of between 60±(20% of60) (i.e., between 48 and 70).

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Thedisclosure illustratively described herein suitably may be practiced inthe absence of any element of elements, limitation or limitations thatis not specifically disclosed herein.

In some embodiments, methods of testing shRNAs targeting HCV IBESsequences are included to identify those sequences having sufficientactivity (e.g., the highest activity among a selected group of suchsequences) to be a candidate for use as a treatment. Testing may alsoinclude screening for shRNAs having undesirable off-target effects, IFNinduction or general cytotoxic effects. Off-target effects include,without limitation, knockdown of nontargeted genes, inhibition ofexpression of non-targeted genes, and competition with natural microRNApathways. Methods of identifying cytotoxic effects are known in the art.

In one embodiment, an shRNA described herein comprises a sequencecomplementary to a sequence of the internal ribosome entry site (IRES)element of hepatitis C virus (HCV).

A dual reporter luciferase plasmid was used in which firefly luciferase(fLuc) expression was dependent on the HCV IRES. Expression of theupstream renilla luciferase is not HCV IRES-dependent and is translatedin a Cap-dependent process. Direct transfection of HCV IRES shRNAsefficiently blocked HCV IBES-mediated flue expression in human 293FT andHuh? cells. Control shRNAs containing a double mutation had little or noeffect on fLuc expression, and shRNAs containing only a single mutationshowed partial inhibition. These shRNAs were also evaluated in a mousemodel where DNA constructs were delivered to cells in the liver byhydrodynamic transfection via the tail vein. The dual luciferaseexpression plasmid, the shRNAs, and secreted alkaline phosphataseplasmid were used to transfect cells in the liver, and the animals wereimaged at time points over 12 to 96 hours. In vivo imaging revealed thatHCV IRES shRNA directly, or alternatively expressed from apolIII-plasmid vector, inhibited HCV IRES-dependent reporter geneexpression; mutant or irrelevant shRNAs had little or no effect. Theseresults indicate that shRNAs, delivered as RNA or expressed from viralor nonviral vectors, are useful as effective antivirals for the controlof HCV and related viruses.

To further investigate the relationship between the RNAi activity andthe structure of synthetic shRNA, multiple shRNAs, fractured shRNA andT-shaped RNA with the guide strand sequence complementary to a sequenceof the HCV IRES and the corresponding synthetic siRNAs comprising thesame sequence were assayed for inhibitory activity, ITN andcytotoxicity. Most of the tested constructs exhibited a high level ofRNAi activity. In general, shRNA with guide strand at the 5′ end of thehairpin was more potent than that with the same guide strand at the 3′end. Structural variants of shRNA with 5′-guide strand were then furtherinvestigated.

The size and sequence of the loop region of the shRNA was alsoinvestigated. The loop can be as small as one nucleotide withoutsignificantly affecting the shRNA activity and does not have a clearupper limit on loop size; generally, a loop is between two to tennucleotides, and is generally a sequence that does not cause unintendedeffects, e.g., by being complementary to non-target gene. However, theshRNA with guide strand at the 5′ end favors a short loop. Specifically,a two-nucleotide loop (5′-UU-3′) provided the shRNA the best potencyamong the shRNAs having ten-, five-, or one-nucleotide loop. The closingbase pair immediately before the loop is important to the short loop(e.g., two-nucleotide loop), but not as important to the long loop(five-nucleotide loop) in order to keep the high functionality of shRNA.Thus, shRNA with “CG clamp” immediately before UU-loop that could serveto strengthen duplex formation gave 4.6-fold lower IC₅₀ than that withan AU base pair (FIG. 5B). In another aspect, the loop can include the3′ part of the guide strand, directly coupled to the 5′ end of thepassenger strand, without affecting the gene knock-down activity of theshRNA. In this case, the loop is between two to eight nucleotides inlength.

A loop structure can also include deoxyribonucleotides, non-nucleotidemonomers and reversible linkages such as S—S bonds, which can be formedby oxidation of —SH groups introduced into nucleotide residues, e.g., asdescribed in (Earnshwaw et al., J. Mol. Biol., 1997, 274:197-212;Sigurdsson et al. Thiol-containing RNA for the study of Structure andFunction of Ribozymes. Methods: A Companion to Methods in Enzymology,1999, 18:71-77).

The length of the duplex in the shRNAs also affects the target genesuppression. The shRNAs that have their passenger strand shortened fromthe 3′ end (to 17 or 16 nucleotides in length) while the guide strand ismaintained as 19-nucleotide have significantly less silencing efficacy.Shortening the passenger strand from the 5′ end (to 17-nucleotide inlength) while maintaining the guide strand at 19 nucleotides does notaffect the shRNA activity. Interestingly, shRNA maintains some activitywith passenger strands as short as 11-nucleotide in length (shortenedfrom the 5′ end) if the guide strand is maintained at 19-nucleotide inlength. shRNAs that have both passenger and guide strands shortened(each 18 nucleotides in length and forming 18 base pairs in the duplex)showed very similar potency compared to the shRNA with 19-base pairedduplex. Single mismatches at certain positions can be introduced intothe duplex region of the shRNA without affecting their potency.

Fractured shRNA and T-shaped RNA were also tested and high levels ofRNAi activity were seen, as shown in Example 6.

Whenever a range is given in the specification, for example, atemperature range, a time range, a percent sequence identity, a sequencecomplementarity range, a length range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure.

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

Examples Example 1 Optimizing shRNA Structure to Improve Potency inTarget Knockdown

To identify shRNA structures with increased potency in target knockdown,three sets of shRNAs (shRNA68, shRNA 72, shRNA 74) with 5′-passengerstrand (right-hand loop) and three with 5′-guide strand (left-hand loop)were chemically synthesized by IDT (Coralvilled, Iowa), resuspended inRNase- and pyrogen-free buffer (Dharmacon) and evaluated; specifically,they were:

shRNA with the structure 5′-passenger strand-5-nt loop-guide strand-3′(SG68, SG72, and SG74)

shRNA with the structure 5′-guide strand-5-nt loop-passenger strand-3′(SG68L, SG72L, and SG74L)

The sequence of these shRNAs are shown in Table 1. shRNA loops areunderlined. Nucleotides forming the 3′ overhang are indicated bylower-case letters.

TABLE 1 Listing of shRNA Sequeuces Targeting HCV IRES Target positionshRNA IC₅₀ Sequence ID# Sequence (5′-3′) on IRES abbrev (pM) SEQ ID NO:1 GCA CGA AUC CUA AAG CUC ACA AUA 346-364 SG68 235.2 UGA GGU UUA GGA UUCGUG Cuu SEQ ID NO: 2 UGA GGU UUA GGA UUC GUG CCA 346-364 SG68L 8.3AUA GCA CGA AUC CUA AAC CUC Auu SEQ ID NO: 3 GUG CAC CAU GAG CAC GAAUCA AUA 335-353 SG72 113.6 AUU CGU GCU CAU GGU GCA Cuu SEQ ID NO: 4 AUUCGU GCU CAU GGU GCA CCA 335-353 SG72L 118.9 AUA GUG CAC CAU GAG CAC GAAUuu SEQ ID NO: 5 CCU AAA CCU CAA AGA AAA ACA AUA 354-372 SG74 95.1 UUUUUC UUU GAG GUU UAG Guu SEQ ID NO: 6 UUU UUC UUU GAG GUU UAG GCA 354-372SG74L 54.6 AUA CCU AAA CCU CAA AGA AAA Auu

Human 293FT (Invitrogen) cells were maintained in DMEM with 10%heat-inactivated fetal bovine serum (Hyclone), supplemented with 2 mML-glutamine and 1 mM sodium pyruvate. The day prior to transfection,cells were seeded at 23,000 cells per well in a 96-well plate, resultingin about 80% cell confluency at the time of transfection. Cells weretransfected with Lipofectamine™ 2000 (Invitrogen, Carlsbad, Calif.)following the manufacturer's instructions. Specifically, synthetic shRNAsamples with indicated concentrations (e.g., 10, 3, 1, 0.3, 0.1, 0.03,0.01 and 0.003 nM),13 ng DNA plasmid pSG154m (which contains an HCV IREStarget sequence and a firefly luciferase reporter sequence), 20 ngpSEAP2-control plasmid (BD Biosciences Clontech, as transfectioncontrols) were mixed with 0.25 μl Lipofectamine 2000 in OptiMem(Invitrogen) and introduced into 293FT cells in triplicate. Forty-eighthours later, the supernatant was removed, heated at 65° C. for 15-30minutes, and 5-10 μl of the supernatant was added to 150 μlp-nitrophenyl phosphate liquid substrate system (pNPP, Sigma). After a30-60 minute incubation at room temperature, samples were read (405 nm)on a Molecular Devices Thermomax microplate reader and quantitated usingSOFTmax software (Molecular Devices). The remaining cells were lysed andluciferase activity was measured using MicroLiimat LB 96P luminometer(Berthold).

The results of these experiments are presented in FIG. 3A-3C. One of thethree shRNAs, sh68, showed nearly a 30-fold higher potency when aleft-hand (instead of right-hand) loop was used. The other two shRNAs,sh72 in particular, did not show a significant difference in potencybetween shRNAs with right- and left-hand loops.

To identify the key attributes associated with the potency enhancementof shRNA with 5′-guide strand, the internal stabilities of shRNAs with5′-passenger strand and shRNAs with 5′-guide strand were calculatedusing the program Oligo 4.0. Only the 19 bases of the 5′ strand werecalculated.

The results of the calculations are presented in FIG. 3D-3F anddemonstrate that the lack of activity increase in sh72 with left-handloop were likely sequence-specific and at least partially due to thedifferences in the internal stability of the 5′ termini. The internalstabilities of the 5′ ends of shRNAs with right- and left-hand loopswere very close to each other in sh68 but not in sh72. It is possiblethat shRNAs with left-hand loops ease the entry of, and foster thepreferential use of, the guide strand since it is at the 5′ end and hasthe 5′ phosphate that is a prerequisite for binding to Dicer and Ago2 inRISC. This advantage disappears when the internal stability of the 5′ends differs significantly between shRNAs with right- and left-handloops.

Example 2 Identifying Optimal Loop Structure and Closing Base Pair ofshRNA

The loop structure of sh19-3 was previously studied (as described inWO2007/032794, specifically incorporated by reference herein). Given thepotency enhancement of SG68L versus SG68 targeting the same IRES regionas sh19-3, the loop structure was re-examined. The effect of variousloop structures on ability to inhibit HCV IRES-mediated reporterexpression in 293FT cells was described in Example 1. The results areshown in FIG. 4. SEAP levels were uniform in all samples, indicatingefficient transfection and the absence of nonspecific inhibitory ortoxic effects, at shRNA concentrations of 0.003 nM to 0.3 nM.

The loop length was first investigated using shRNAs targeting the sameIRES region as siRNA 19-3 (si19-3). The structure of shRNAs was 5′-guidestrand-loop-passenger strand-3′ (left-hand loop). The duplex length was19-base pair with a UU-overhang at the 3′ end of the passenger strand.The sequences of shRNAs with various lengths of loop are listed in Table2. shRNA loops are underlined. Nucleotides of the 3′ overhang areindicated by lower-case.

The dose response of shRNAs with a 10-nucleotide loop (SG113,5′-CUUCCUGUCA-3′), 5-nucleotide loop (SG68L, 5′-CAAUA-3′), 2-nucleotideloop (SG142, 5′-UU-3′) and 1-nucleotide loop (SG114, 5′-U-3′) wereexamined and the results are shown in FIG. 4A. Unlike the results withshRNAs having 5′-passenger strands (right-hand loops) (Vlassov et al.,Oligonucleotides 17:223-236, 2007), the shRNA with 5′-guide strands(left-hand loops) had higher potencies when the loop size was very small(1 or 2 nt) than when it was larger (5 or 10 nt; FIG. 4A). The loopsequence likely doesn't affect the shRNA activity since 3 different loopsequences were used, including one derived from a miR-23 microRNA loopstructure. The IC₅₀ of SG142 (left-hand 5′-UU-3′ loop) was as low as 4.6pM - more potent than siRNA 19-3, which targets the same region. TheshRNAs with right-hand loops have A:U as the base pair immediatelyadjacent to the loop, and those with left-hand loops have a C:G pair atthat same position. These pairs may not in fact be base paired torelieve potential strain from a 2-nt loop. If that is the case, the C:Gbase pair of SG118 may participate in a tetraloop structure (CUUG) thatis more stable than the 4-nt loop AUUU of SG103. Interestingly, thetetraloop-forming SG118 had the lowest 10₅₀ (FIG. 4B, SG118). Thesequences of the duplex region are the same among these shRNAs and arelisted in Table 2.

To make the loop resistant to cleavage by endoribonucleases, the2′-deoxy dinucleotide TT was tested as the loop. No significant loss ofactivity (FIG. 4C) was detected upon replacement of the loop sequence UU(SG142), with TT (SG112), indicating that deoxy-substitution could beapplied to the loop for ribonuclease resistance without sacrificingfunctional activity. It also suggests that neither Dicer nor any otherendoribonuclease was involved in the processing of 19-bp shRNA, sincedeoxyribonucleotides cannot be cleaved by RNases, except perhaps at the5′ side of the dinucleotide.

The sequences of these shRNAs are shown in Table 2. shRNA loops areunderlined. Nucleotides of the 3′ overhangs are indicated by lower-caseletters.

TABLE 2 Listing of shRNA Sequences with Various Loop Structure andClosing Base Pairs Target position shRNA IC₅₀ Sequence ID# Sequence(5′-3′) on IRES abbrev (pM) SEQ ID NO: 7 UGA GGU UUA GGA UUC GUG CCU UCC346-364 SG1113 36.3 UGU CAG CAC GAA UCC UAA ACC UCA uu SEQ ID NO: 2 UGAGGU UUA GGA UUC GUG CCA AUA 346-364 SG68L 17.5 GCA CGA AUC CUA AAC CUCAuu SEQ ID NO: 8 UGA GGU UUA GGA UUC GUG CUU GCA 346-364 SG142 8.7 CGAAUC CUA AAC CUC Auu SEQ ID NO: 9 UGA GGU UUA GGA UUC GUG CUG CAC 346-364SG114 10.3 GAA UCC UAA ACC UCA uu SEQ ID NO: 3 GUG CAC CAU GAG CAC GAAUCA AUA 335-353 SG72 94.1 AUU CGU GCU CAU GGU GCA Cuu SEQ ID NO: 4 AUUCGU GCU CAU GGU GCA CCA AUA 335-353 SG72L 81.3 GUG CAC CAU GAG CAC GAAUuu SEQ ID NO: 10 AUU CGU GCU CAU GGU GCA CUU GUG 335-353 SG118 72.7 CACCAU GAG CAC GAA Uuu SEQ ID NO: 11 GUG CAC CAU GAG CAC GAA UUU AUU335-353 SG103 405.4 CGU GCU CAU GGU GCA Cuu SEQ ID NO: 12 UGA GGU UUAGGA UUC GUG CTT GCA 346-364 SG112 15.4 CGA AUC CUA AAC CUC Auu

Example 3 Investigating the Relationship Between Efficacy and shRNAOverhang and Duplex Length

Vlassov et al. found that the presence of a 3′-UU overhang increases theefficacy of 19-bp shRNAs (Vlassov, et al. 2007). The shRNAs used in thisprevious study had right-hand loops (5′-passenger strand-loop-guidestrand-3′). Thus, the UU overhang at the 3′ end of the guide strand mayfacilitate the binding of the Ago PAZ domain to the 3′-UU overhang ofthe guide strand. Since the shRNA with left-hand loop (5′-guidestrand-loop-passenger strand-3′) showed better target gene suppression,the effect of a UU overhang at the 3′ end of the passenger strand onshRNA efficacy was examined As shown in FIG. 5A, an shRNA lacking the3′-UU sequence (SG105) and an shRNA having a deoxyribonucleotide 3′-TTas overhang (SG111) had very similar potency to the corresponding shRNAwith 3′-UU overhang.

The effect of stem length on shRNA activity was also investigated. Asshown in FIG. 5B, shortening the passenger strand to 17- or 16-nt whilemaintaining the length of the guide strand at 19-nt significantlyreduced gene silencing activity (SG115 and SG116). However, shorteningboth strands to 18-nt in length did not have a significant impact onpotency (SG117). Surprisingly, an shRNA with a stem as short as 16-bp(SG119 with 19-nt guide strand and 17-nt passenger strand) showedsimilar inhibition to one having a 17-bp stem (SG117) (FIG. 5C).However, shRNAs with a stem of 15-bp (SG131) started to show reducedinhibitive activity (FIG. 6A). This indicates that high silencingactivity requires a duplex length of 16-bp or higher.

To test an extreme case, the silencing by an shRNA (SG120) with an 11-bpduplex and an 8-nt loop (a 19-nt guide strand directly linked to an11-nt passenger strand) was measured (FIG. 5C). Although the potency waslower compared to the others, the short RNA hairpin still had an IC₅₀ ofless than 200 pM. Similarly, an shRNA (SG134) with a 13-bp duplex and a6-nt loop (a 19-nt guide strand directly linked to a 13-nt passengerstrand) gave the same level of target knockdown (FIG. 6B). A furthershortening of the passenger strand to 10-nt diminished the silencingsignificantly. Changing the 11-nt passenger strand from 3′ end to 5′ endof the hairpin (SG135) abolished the silencing (FIG. 6B).

To examine whether the overhang and stem length effects'weresequence-specific, two target sequences other than the one used in FIGS.5 and 6 were tested (FIGS. 7A and 7B). Depletion of the 3′-UU overhangdid not significantly reduce the target silencing of the hairpins.Shortening the stem to 18-bp (SG139) or 16-bp (SG136) reduced thehairpins' target knock down capability, indicating this stern shorteningstrategy has some degree of sequence dependency.

The sequences of these shRNAs are shown in Table 3. shRNA loops areunderlined. Nucleotides of the 3′ overhangs are indicated by lower-case.

TABLE 3 Listing of shRNA Sequences with Various Duplex Structures Targetposition shRNA IC₅₀ Sequence ID# Sequence (5′-3′) on IRES abbrev (pM)SEQ ID NO: 13 UGA GGU UUA GGA UUC GUG CUU GCA 346-364 SG105 4.2- CGA AUCCUA AAC CUC A 10.9 SEQ ID NO: 14 UGA GGU UUA GGA UUC GUG CUU GCA 346-364SG111 16.2 CGA AUC CUA AAC CUC Att SEQ ID NO: 8 UGA GGU UUA GGA UUC GUGCUU GCA 346-364 SG142 5.7- CGA AUC CUA AAC CUC Auu 10.6 SEQ ID NO: 15UGA GGU UUA GGA UUC GUG CUU GCA 346-364 SG115 26.6 CGA AUC CUA AAC CUSEQ ID NO: 16 UGA GGU UUA GGA UUC GUG CUU GCA 346-364 SG116 1007.4 CGAAUC CUA AAC C SEQ ID NO: 17 UGA GGU UUA GGA UUC GUG UUC ACG 346-364SG117 15-19.9 AAU CCU AAA CCU CA SEQ ID NO: 18 UGA GGU UUA GGA UUCGUG CAC GAA 346-364 SG119 3.7- UCC UAA ACC UCA 26.8 SEQ ID NO: 19 UGAGGU UUA GGA UUC GUG CCC UAA 346-364 SG120 87.9- ACC UCA 158.0 SEQ ID NO:20 UGA GGU UUA GGA UUC GUG CUU GCA 346-364 SG110 7.3 CGU AUC CUA AAC CUCAuu SEQ ID NO: 21 UGA GGU UUA GGA UUC GUU CGA AUC 346-364 SG131 8.6 CUAAAC CUC A SEQ ID NO: 22 UGA GGU UUA GGA UUC UUG AAU CCU 346-364 SG13226.0 AAA CCU CA SEQ ID NO: 23 UGA GGU UUA GGA UUC GUG AC UAA 346-364SG133 698.3 ACC UCA SEQ ID NO: 24 UGA GGU UUA GGA UUC GUG CAU GG 346-364SG134 121.9 UAA ACC UCA SEQ ID NO: 25 GCA CGA AUC CUU GAG GUU UAG GAU346-364 SG135 4,863.8 UCG UGC SEQ ID NO: 10 AUU CGU GCU CAU GGU GCACUU GUG 335-353 SG118 51.9 CAC CAU GAG CAC GAA Uuu SEQ ID NO: 26 AUU CGUGCU CAU GGU GCA CGC ACC 335-353 SG136 393.1 AUG AGC ACG AAU SEQ ID NO:27 AUU CGU GCU CAU GGU GCA CUU GUG 335-353 SG137 129.0 CAC CAU GAG CACGAA U SEQ ID NO: 28 UUU UUC UUU GAG GUU UAG GUU CCU 354-372 SG108 21.0AAA CCU CAA AGA AAA Auu SEQ ID NO: 29 UUU UUC UUU GAG GUU UAG GUU CCU354-372 SG138 65.9 AAA CCU CAA AGA AAA A SEQ ID NO: 30 UUU UUC UUU GAGGUU UA GUU CU 354-372 SG139 1,343.0 AAA CCU CAA AGA AAA Auu

Example 4 Investigating the Effect of the Position and Type of SingleMismatch in 19-bp shRNA

It has been shown that both guide and passenger strands of siRNA can beincorporated into RISC complex. This may be applicable to guide andpassenger strands of shRNA. Since the selection of a passenger strand byAgo could induce unwanted effects if the passenger strand (the seedregion in particular) were complementary to the coding region or 3′-UTRof messenger RNA, single mutations were made at positions 4 to 7 from 5′end of the passenger strand.

As shown in FIGS. 8A and 8B, these mutations did not significantlyaffect the potency of shRNA with both strands 19-nucleotide in length,3′ UU overhang and UU-loop. In addition, this maintenance of activitywith single mismatch did not appear to be unrelated to the type ofmismatch (e.g., U-U (SG110)=U-C (SG126)), suggesting that this trait issequence-independent.

The sequences of these shRNAs are shown in Table 4. shRNA loops areunderlined. Nucleotides of the 3′ overhangs are indicated by lower-case.Mismatched nucleotide is indicated in italics.

TABLE 4 Listing of shRNA Sequences with Single Mismatch in the PassengerStrand Target position shRNA IC₅₀ Sequence ID# Sequence (5′-3′) on IRESabbrev (pM) SEQ ID NO: 8 UGA GGU UUA GGA UUC GUG CUU GCA 346-364 SG1424.6-8.7 CGA AUC CUA AAC CUC Auu SEQ ID NO: 20 UGA GGU UUA GGA UUC GUGCUU GCA 346-364 SG110 7.3 CGU AUC CUA AAC CUC Auu SEQ ID NO: 31 UGA GGUUUA GGA UUC GUG CUU GCA 346-364 SG126 3.5 CGC AUC CUA AAC CUC Auu SEQ IDNO: 32 UGA GGU UUA GGA UUC GUG CUU GCA 346-364 SG127 3.5 CGA CUC CUA AACCUC Auu SEQ ID NO: 33 UGA GGU UUA GGA UUC GUG CUU GCA 346-364 SG128 3.6CCA AUC CUA AAC CUC Auu SEQ ID NO: 34 UGA GGU UUA GGA UUC GUG CUU GCA346-364 SG129 4.2 GGA AUC CUA AAC CUC Auu

Example 5 Investigating the Effect of Monomer and Dimer on shRNAEfficacy

The shRNAs synthesized and HPLC-purified by IDT were found to comprisethree major species in native polyacrylamide gels: monomer, dimer andtrimer. In contrast, under denaturing conditions (12% polyacrylamide gelcontaining 8 M urea and 20% formamide), the mixture showed only a singlemajor band. When the shRNAs were heated to 95° C. for 4 minutes andquickly cooled in an ice bath, this mixture of monomer, dimer and trimercould be largely transformed to monomer (FIG. 9B and FIG. 10B), andmonomer shRNAs remained strong inhibitors of IRES-dependent luciferaseexpression in 293FT cells, albeit slightly weaker than the mixture (FIG.9A). When the shRNA was shortened to 16-nucleotide (SG119) and the dimerof this molecule formed a perfect duplex without bulge or mismatch, themonomers had better efficacy than monomer-dimer mixtures (FIG. 10A).This was probably due to the fact that dimers had an inefficientcleavage or various cleavage products by Dicer. However, when the shRNAwas extremely short (11 base pairs with the rest of guide strand as loop(SG120)), the hairpin was more active in mixture, probably in dimerform, than in monomer only form (FIG. 10A). But even in pure monomerform, SG120 reduced the target gene expression with high efficiency,with IC₅₀ of 98.6 pM.

Example 6 Identifying Optimal shRNA Configuraton with EnhancedFunctionality

To identify optimal shRNA configurations that have enhanced geneknockdown, 3 shRNAs targeting the same region of IRES were synthesizedand evaluated.

a. SG68L: 5′-guide strand-CAAUA (loop)-passenger strand-3′

b. SG146: Annealed products from two RNA molecules. SG146-1, 5′-guidestrand-CAAUA (loop)-partial passenger strand-3′; SG146-2, 5′-partialpassenger strand that is complementary to guide strand

c. SG142: 5′-guide strand-UU (loop)-passenger strand-3′ shRNA sampleswith different concentrations (0.3, 0.1, 0.03, 0.01 and 0.003 nM) and 13μg DNA plasmid pSG154m (containing target sequence HCV IRES and reportersequence firefly luciferase) and pUC19 (made up the same amount ofnucleic acids) were mixed with 0.25 ml Lipofectamine 2000 (Invitrogen)in OptiMem (Invitrogen) and introduced into 293FT cells (23,000 cellsper well in 96-well plates). The knockdown of firefly luciferase geneexpression was measured by a luminometer at 48 hrs.

The results of these experiments are presented in FIG. 11 anddemonstrate: 1) shRNA with 2-nucleotide loop (SG142) was slightly morepotent than shRNA with 5-nucleotide loop (SG68L); and 2) shRNA annealedfrom two molecules (SG146) had good knockdown functionality.

The sequence sof these shRNAs are shown in Table 5. shRNA loops areunderlined. Nucleotides of the 3′ overhangs are indicated by lower-case.

TABLE 5 Listing of shRNA Sequences with Various Loop Structure andClosing Base Pairs Target position shRNA IC₅₀ Sequence ID# Sequence(5′-3′) on IRES abbrev (pM) SEQ ID NO: 2 UGA GGU UUA GGA UUC GUG CCA AUA346-364 SG68L 26.6 GCA CGA AUC CUA AAC CUC Auu SEQ ID NO: 8 UGA GGU UUAGGA UUC GUG CUU GCA 346-364 SG142 11.5 CGA AUC CUA AAC CUC Auu SEQ IDNO: 35 UGA GGU UUA GGA UUC GUG CCA AUA 346-364 SG146- 61 GCA C 1 SEQ IDNO: 36 GAA UCC UAA ACC UCA uu SG146- 2 SEQ ID NO: 37 GCA CGA AUC CUA AACCUC AAA GCA 346-364 SG102- UGC UCC 1 SEQ ID NO: 38 ACC GUG GUC UUU GAGGUU UAG GAU SG102- UCG UGC UU 2 SEQ ID NO: 39 GGA GCA UGA CCA CGG USG102- 3

Example 7 Investigating Silencing Effect of shRNAs in Hepatoma Cell line

These experiments were performed similarly to those described in Example1, except that the cell line was changed to the hepatocarcinoma cellline Huh? (FIG. 12). Three shRNAs with various concentrations werecompared, including SG68, SG68L and negative control SG101 (sequence:5′-CGU GCU UAG GAU UUG GAG UCA AUA ACU CCA AAU CCU AAG CAC GUU-3′). Noreduction of firefly luciferase was found in cells transfected withSG101. Consistent with the results in 293FT cell, SG68L is much morepotent than SG68 in silencing target gene expression in hepatocarcinomacells.

Example 8 High Efficacy of shRNAs are not due to IFN Response orCytotoxicity

Some siRNAs or shRNAs induce an IFN response and/or off-target effects.To investigate whether the shRNAs tested above had these unwantedfeatures, the IFN-responsive gene OAS1 was measured after shRNAtransfection. Specifically, human PBMCs were prepared from buffy coatsby density gradient centrifugation, washed, and then resuspended in RPMI1640 containing 10% heat-inactivated fetal calf serum. The cells werecultured at 5×10⁵ cells per well in 24-well plates and then transfectedfor 24 hours with shRNAs (20 nM, about 170 to 180 ng)/DOTAP (1.5 μl,Roche) complexes. The cells were then lysed in Trizol (Invitrogen) andtotal RNA was extracted according to the manufacturer's instructions.qRT-PCR was done using High-Capacity cDNA Reverse Transcription Kits,TaqMan Universal PCR Master Mix, OAS1 (Hs00242943_ml) and GAPDH(Hs99999905_ml) Taqman probe and Fast 7500 real time PCR machine(Applied Biosystem) according to the manufacturer's instructions.

As shown in FIG. 13A, the positive control, Poly (I:C) 18 ng per well(equivalent to the amount of shRNA per well) transfected with DOTAPyielded a 10.6 fold increase of OAS 1 expression compared to untreatednegative control. However, none of the shRNAs tested resulted in anincrease of OAS1, indicating that no IFN was induced in human PBMC.

Cytotoxicity was also tested by measuring3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)incorporation in human hepatocarcinoma cell line (Huh7) after shRNAtransfection. Huh7 cells seeded in 96-well plate (13,000 cells/well) thenight before were transfected with 10 nM shRNA together with target DNAusing Lipofectamine 2000 (LF2K, Invitrogen). Poly (I:C) (33 ng) was usedas positive control. 48 hours later, 16 ul/well of MTT (Sigma) inHepes-buffered saline at 5 mg/ml was added to the culture. The cellswere further incubated for 3 hours before being lysed with acidicisopropanol (0.04N HCl). The dissolved formazan level was measured by aMolecular Devices Thermomax microplate reader and quantitated usingSOFTmax software (Molecular Devices) with a test wavelength of 570 nmand a reference wavelength of 650 nm.

As shown in FIG. 13B, no significant cell death relative to the negativecontrol, plasmid DNA in lipofectamine, was found. The positive control,poly(I:C) in lipofectamine, induced more than 50% cell death. Overall,the shRNAs tested above showed no IFN and no significant cytotoxicity inhuman PBMC and hepatocarcinoma cells.

Example 9 shRNAs with Duplex Length of 19 Base Pairs or Less are notDicer Substrates

To investigate whether shRNAs with duplex lengths of 19 base pairs orless are able to be cleaved by Dicer, several shRNAs were selected forin vitro Dicer cleavage analysis. An shRNA (shl) with duplex length of25 base pairs (5′-GGGAGCACGAAUCCUAAACCUCAAAGACUUCCUGUCAUCUUUGAGGUUUAGGAUUCGUGCUCUU-3′) (SEQ ID NO:40) was used as positive control.Specifically, all synthetic shRNAs were dissolved to 5μM finalconcentration in buffer containing 20 mM KCl, 6 mM HEPES-KOH (pH 7.5),0.2 mM MgCl₂. To ensure that all molecules formed hairpin monomers,shRNAs were heated to 95° C. for 4 min and then were transferredimmediately to an ice-water bath to cool for ˜10-20 min before furtheruse. 8 pmol of each shRNA was incubated in a 10 μL reaction in thepresence of 1U of recombinant dicer enzyme (Stratagene, Catalog #240100)and 1× buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 8), and 2.5 mMMgCl₂ for 18 hours at 37° C. Control reactions that contained each shRNAbut lacked dicer enzyme were incubated in parallel.

Samples were analyzed by both 10% non-denaturing PAGE (FIG. 14A) and 12%denaturing PAGE (8M urea/20% formamide) (FIG. 14B) and were stained withSYBR Gold (Invitrogen). As shown in FIG. 14, the shl RNA that containeda duplex of 25 base pairs in length, a 10-nucleotide loop and 5′- and3′- overhangs was cleaved by Dicer to generate a product with a sizebetween 20 to 25 base pairs. All other shRNAs tested, including threewith a stem of 19 base pairs and a hairpin of 2 or 5 nucleotides inlengths and one with a stem. of 18 base pairs and a haipin of 2nucleotides, did not show any cleavage products in both non-denaturingand denaturing PAGEs. These results indicate that short hairpins with aduplex length of 19 base pairs or less are not Dicer substrates.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An RNA molecule comprising: a first RNA sequence consisting of 15nucleotides to 30 nucleotides, wherein the first sequence is at leastpartially complementary to a target nucleotide sequence; a second RNAsequence comprising a sequence that is at least partially complementaryto at least a portion of the first sequence, the second sequence havinga length of between 10 nucleotides and 1 fewer nucleotide than thelength of the first sequence; optionally a loop sequence positionedbetween the first sequence and the second sequence, the loop consistingof 1 to 100 nucleotides; and optionally a nucleotide overhang consistingof 1 to 2 nucleotides.
 2. (canceled)
 3. The RNA molecule of claim 5,wherein: the first RNA sequence consists of 16 nucleotides to 18nucleotides; and the second RNA sequence has the same number ofnucleotides as the first sequence.
 4. The RNA molecule of claim 1,wherein: the first RNA sequence consists of 19 nucleotides; and thesecond RNA sequence consisting consists of 17 nucleotides or 18nucleotides.
 5. An RNA molecule comprising: a first RNA sequenceconsisting of 15 nucleotides to 30 nucleotides, wherein the firstsequence is at least partially complementary to a target nucleotidesequence; a second RNA sequence comprising a sequence that is at leastpartially complementary to at least a portion of the first sequence, thesecond sequence having the same or fewer number of nucleotides as thefirst sequence; optionally a loop sequence positioned between the firstsequence and the second sequence, the loop consisting of 1 to 2nucleotide and/or non-nucleotide moieties; and optionally a nucleotideoverhang consisting of 1 to 2 nucleotides; wherein when the first RNAsequence and the second RNA sequence are 19 nucleotides, the optionalloop sequence is 1 nucleotide.
 6. The RNA molecule claim 5, wherein thesecond sequence comprises a sequence that is at least 85% complementaryto the portion of the first sequence.
 7. The RNA molecule of claim 6,wherein the portion of the first sequence is at the 5′ end of the firstsequence.
 8. The RNA molecule of claim 7, wherein the sequence that isat least 85% complementary to the portion of the first sequence is asequence at the 3′ end of the second sequence.
 9. (canceled)
 10. The RNAmolecule of claim 36, wherein the loop comprises at least onenon-nucleotide moiety.
 11. The RNA molecule of claim 37, wherein thenucleotide overhang is on the 3′ end of the RNA molecule.
 12. The RNAmolecule of claim 5, wherein the RNA molecule comprises 1 or 2mismatches between the first sequence and the second sequence. 13-14.(canceled)
 15. The RNA molecule of claim 5, wherein the molecule iscapable of inhibiting expression of the target nucleotide sequence. 16.The RNA molecule of claim 5, wherein the target nucleotide sequence is aviral sequence.
 17. The RNA molecule of claim 16, wherein the viralsequence is a hepatitis C viral sequence.
 18. The RNA molecule of claim17, wherein the hepatitis C viral sequence is a sequence within theinternal ribosome entry site (IRES) sequence of hepatitis C virus.19-21. (canceled)
 22. A DNA sequence comprising a sequence encoding theRNA of claim
 5. 23. The DNA sequence of claim 22, wherein the DNAsequence is selected from: an expression vector and a retroviral vector.24-33. (canceled)
 34. The RNA molecule of claim 1, wherein the RNAmolecule is selected from any one of SEQ ID NOs: 2, 6, 7 and
 35. 35. TheRNA molecule of claim 5, wherein the RNA molecule is selected from anyone of SEQ ID NOs: 8, 9, 12, 13, 14, 15, 17, 18, 20, 21, 22, 28, 31, 32,33, and
 34. 36. The RNA molecule of claim 5, wherein the RNA moleculecomprises the optional loop sequence.
 37. The RNA molecule of claim 5,wherein the RNA molecule comprises the optional nucleotide overhang.